PROJECT SUMMARY/ABSTRACT Plant natural products (NPs) are a critical source of clinically approved drugs and dietary nutrients, yet very few complete biosynthetic pathways have been characterized. As a consequence, many complex plant natural product scaffolds are currently still isolated from the producing plant or plant cell culture and then converted to a clinically-used drug by semisynthetic routes (e.g. etoposide, digoxin, morphine, vinblastine, and paclitaxel ? all on the 2015 WHO list of essential medicines). Lack of information regarding their biosynthetic pathways severely limits the use of promising new approaches to produce plant molecules in heterologous hosts (e.g. yeast strains that make artemisinin), as well as the intriguing possibility of engineering the biosynthetic pathways to access analogs and non-natural derivatives with greater efficacy. Even less is known about pathways that could be the target of engineering or breeding efforts in edible plants to improve nutrient content. Given the critical role of plant natural products in human health and utility of biosynthetic genes, we propose here the development and application of a broadly generalizable platform to accelerate the discovery and engineering of key plant natural product pathways. Classically, the discovery of plant pathways has been slower and more painstaking than bacterial pathways; however, we have recently shown that combining three technologies greatly accelerates rapid plant pathway discovery that we will further expand as part of this effort: (1) rapid combinatorial testing of enzymes in a tobacco heterologous host, (2) transcriptional profiling and co-expression analysis to identify pathway genes, and (3) untargeted metabolomics as an in-line analytical tool. This approach enabled the discovery of six enzymes that complete the ten step pathway to the etoposide aglycone from the unsequenced medicinal plant Podophyllum in a matter of months (previous work to elucidate the first four steps was reported over ~ a dozen years). In this proposal we have prioritized pathways for clinically used NPs (etoposide), molecules abundant in edible plants (Brassica indolic phytoalexins and falcarindiol from carrot and tomato), or clinical candidates whose assessment would be enabled by the ability to generate the native compound or analogs (limonoids). These compounds represent a diverse set of NP classes and will be used to demonstrate the broad utility of our discovery approach. In each case we will also focus on development of tobacco as a novel production platform. A major outcome of this work will be sets of biosynthetic genes that can be used to engineer heterologous hosts to make plant NPs and analogs with potent biological activity.